The major objective of the proposed research is to conduct in-depth studies on the mode of action of several compounds representing three groups of insecticide synergists (substituted methylenedioxygenzenes, 1,2,3-benzothiadiazoles and imidazoles) known to act by inhibition of microsomal mixed-function oxidation. In vitro studies will be carried out with microsomes from control and induced (phenobarbital, 3-methylcholanthrene) rat livers and midgut tissues from control and induced (pentamethylbenzene) armyworm (Spodoptera eridania) larvae. Emphasis will be placed on studying the formation of irreversible, inhibitory product adducts with cytochrome P-450 in NADPH-reduced microsomes and on the possibility that the synergists may interact with specific types or pools of cytochrome P-450. The formation of product adducts will be studied by difference spectroscopy and/or the binding of appropriately labeled synergists. The properties of any residual "non-reactive" cytochrome P-450 will be studied by enzymatic assay (epoxidation, hydroxylation and N-demethylation) and a variety of spectral techniques. Equilibrium dialysis will be used to study the binding of labeled 1-phenylimidazole to microsomes and to evaluate this ligand as a potential tool for qualitative and quantitative measurements of different types of cytochrome P-450 in intact microsomes. Studies with rats and armyworm larvae will be carried out to determine the formation and reversibility of synergist-cytochrome P-450 complexes in vivo and to evaluate the effects of these complexes on catalytic activity.